7-O-Carbamoylheptose derivatives, process for their production and their use in treating pseudomonas infections

ABSTRACT

The invention describes 7-O-carbamoyl heptose derivatives of general formula (I) in which R is the substituent R 1  or a group of general formula (II) in which R 1  is a hydrogen atom, a methyl group or a suitable linker substituent suitable for a covalent coupling, a process for their production and their use in producing reagents and compositions for the diagnosis and therapy of pseudomona infections in humans and animals, and a screening process for their detection in Gram-negative bacteria. ##STR1##

This case is filed under 35 USC 371 as the U.S. atage of PCT/EP95/02780filed Jul. 14, 1995.

AREA OF THE INVENTION

The present invention relates to 7-O-carbamoylheptose derivatives, to aprocess for their production and to their use for the production ofreagents and compositions for the diagnosis and therapy of pseudomonasinfections in humans and animals, and to a screening process for theirdetermination in Gram-negative bacteria.

BACKGROUND OF THE INVENTION

Bacteria of the Pseudomonadaceae family are Gram-negative organismswhich occur ubiquitously and whose pathogenicity for humans is normallyvery weakly developed. P. aeruginosa, by contrast, is a human-pathogenicspecies and occurs frequently in wound infections and there especiallyas secondary infection in cases of higher-degree burns of the skin, andin cases of suppurative otitis media. In immunocompromised patients andin cases of cystic fibrosis it is particularly the antibiotic-resistantpseudomonads which are of outstanding medical importance McManus et al.,J. Trauma 21 (1981) 753-763. Bodey et al., Rev. Inf. Dis., 5 (1983),279-313, Winkler et al., Klin. Wochenschrift, 63 (1985) 490-498!.

In terms of taxonomy, pseudomonads comprise a very heterogeneous family.Their species-related heterogeneity represents a considerable impedimentto the medical diagnosis and therapy of pseudomonas infections. This iswhy it was only recently proposed that this family of Pseudomonadaceaebe divided into five different subgroups N. J. Palleroni, Antonie VanLeeuwenhoek, 64 (1993) 231-251!. The first group (RNA group 1) includesP. aeruginosa, P. fluorescens and P. putida. The second group (RNA group2) comprises the pseudomonads which are pathogenic for plants andanimals (for example P. plantarii) and is now referred to asburkholderia. Finally, a distinction is also made between comamonas(group 3) and the purple bacteria (group 4) which continue to bereferred to as "pseudomonas" (for example P. diminuta, P. vesicularis)and lastly also xanthomonas (RNA group 5).

Beyond this, pseudomonads are also of biomedical interest for otherreasons. They are extremely resistant to antibiotics A. M. Kropinski etal., Antimicrob. Agents Chemother., 36 (1985) 58-73!. There is evidencethat this antibiotic resistance is associated with the structure of thecell wall membrane, that is to say with a high density of negativelycharged phosphate groups on molecules in the outer cell wall membranesR. E. W. Hancock et al., in: Bacterial Cell Wall, J. M. Ghuysen and R.Hakenbeck (eds.), Elsevier Amsterdam, 1994, 263-279). Such surfacestructures are in all Gram-negative bacteria essentially integralproteins of the cell membrane (OMP, porins) and the lipopolysaccharide(LPS, endotoxin). These molecules represent antigens which show highgenus-, species- and subspecies-specific immunogenicity and, during thecourse of an infection, induce serotype-specific antibodies which may beof great importance to both diagnosis and therapy.

Over the course of the last decade it has been possible to acquireconsiderable knowledge about the serological and biological propertiesof these antigens. There has also been intensive study and elucidationof the LPS structures and there particularly of their immunogenic outercomponents (O chains) of the 17 serotypes now known. There has beencomplete chemical analysis of all P. aeruginosa O chains, and some ofthem have also been synthesized and classified serologically intovarious immunotypes (Fischer) and serotypes (Lanyi, Habs) N. K.Kochetkov and Yu. A. Knirel, Sov. Sci. Rev. B. Chem. 13 (1989) 1-101!.Based on this structural knowledge, various P. aeruginosa serotypingkits with monoclonal antibodies have been produced and are stillcommercially available. The disadvantage of these antibody testcocktails is that they detect only the known antibodies, but allmonoclonal O-specific antibodies are necessary in order to detect all Otypes.

Besides the antigenic assay processes which have been known for a longtime for the immunologically highly specific O chains, there hasrecently been development of cross-reacting monoclonal antibodies whoseepitope is located not in the highly variable O chain of thelipopolysaccharide but in the less variable core oligosaccharide. Sincethese structures make essential contributions to the function of theouter cell membrane, core oligosaccharides are regarded rather asconservative structural elements of the immunogenic LPS, against whichit might be possible to produce broadly cross-protecting antibodies.This has recently been experimentally demonstrated for the first time. Amouse monoclonal antibody (MAb) whose specificity was directed againstthe core region showed broad cross-reactivity and broad cross-protectionfor all five Escherichia coli (R1, R2, R3, R4, K-12) and for theSalmonella minnesota core oligosaccharide structure, irrespective of thestructure of the O chain of the particular LPS Di Padova et al., Infect.Immun. 61 (1993) 3863-3872!. This MAb (WN1 222-5) of the IgG2a classconfers broad cross-protection against S. minnesota and E. coliendotoxin but not against P. aeruginosa or Klebsiella pneumoniae. Thereason for this is ascribed to the different core oligosaccharidestructure. The core oligosaccharide structures of P. aeruginosa and K.pneumoniae have hitherto been analysed only incompletely or aresubstantially unknown.

In a study in which various rough form mutants of P. aeruginosa wereproduced and their core oligosaccharide was chemically investigated P.S. N. Rowe & P. M. Meadow, Eur. J. Biochem. 132 (1983) 329-337!, thefirst proposed structures of the core region were published. Onestructural pecularity was the presence of the amino acid alanine (Ala)in the outer core oligosaccharide of all the P. aeruginosa rough formmutants investigated. This proposed structure was not revised andimproved until ten years later, on a P. aeruginosa mutant R5 (Habs 06)!by ¹ H-NMR spectroscopy E. Altman et al., Int. Carb. Conference, Paris1992, E. Altman et al., 2nd IES Conference, Vienna 1992!.

However, very recent investigations in our laboratory have revealed thateven this structure of the core oligosaccharide of the deep roughmutants R5 (Habs 06(! derived from the rough form mutants PAClR of P.aeruginosa is still incomplete. It was initially known that this coreregion is extensively phosphorylated. On the other hand, the degradationmethods and analytical processes used by E. Altman et al. wereessentially unsuitable to allow the7-O-carbamoyl-L-glycero-D-manno-heptopyranose which is described indetail for the first time according to the present invention to beidentified and analyzed by spectrometry (¹ H-NMR).

SUMMARY OF THE INVENTION

The inventors have investigated the chemical structure of thelipopolysaccharide of pseudomonads with the aim of providing specificmono- and disaccharides which can be employed as serological markers inthe diagnosis and therapy of pseudomonas infections.

In these investigations it was possible to analyze and completelycharacterize the chemical structure of a previously unknown7-O-carbamoyl-L-glycero-D-manno-heptopyranose in the coreoligosaccharide of the LPS of pseudomonads of RNA group 1. It has notbeen possible to find this heptopyranose in other Gram-negative bacteriaapart from pseudomonads of RNA group 1 (for example in allEnterobacteriaceae).

The invention therefore relates to 7-O-carbamoylheptose derivatives ofthe general formula (I) ##STR2## in which R is the substituent R¹ or agroup of the general formula (II) ##STR3## in which R¹ is a hydrogenatom, a methyl group or a linker substituent suitable for a covalentcoupling.

The invention furthermore relates to a process for the production of the7-O-carbamoylheptose derivatives which is characterized in that alipopolysaccharide of pseudomonads of RNA group 1 is created withhydrofluoric acid, the resulting product is dialysed against water untila neutral pH is reached, the lipopolysaccharide which has beendephosphorylated in this way is methanolyzed, and subsequently apermethylation or peracetylation is carried out, the resulting7-O-carbamoylheptose derivatives are fractionated by liquidchromatography (checking the purity by gas-liquid chromatography orcombined gas-liquid chromatography/mass spectrometry) and, if required,the substituent R¹ is introduced into the resulting 7-O-carbamoylheptosederivatives by a process known per se.

The invention furthermore relates to a screening process for determiningthe 7-O-carbamoylheptose derivatives in Gram-negative bacteria, which ischaracterized in that intact bacteria are treated, without previousremoval of the lipopolysaccharide, with hydrofluoric acid, thenhydrolyzed or methanolyzed to liberate the heptose derivatives, andsubsequently a permethylation is carried out and the permethylatedproduct is analyzed by gas-liquid chromatography or combined gas-liquidchromatography/mass spectrometry.

The 7-O-carbamoylheptose derivatives of the general formula (I) can beused to produce reagents and compositions for the diagnosis and therapyof pseudomonas infections in humans and animals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The linker substituent indicated by R¹ in the general formula (II) ispreferably a cysteamine residue, an allyl group or a straight-chain orbranched-chain C₁₋₁₈ -alkyl group which may contain a terminal hydroxyl,amino, acyl, carboxyl or allyl group. R¹ is preferably a hydrogen atomor a methyl group.

The present invention has succeeded in making the heptose region in thecore oligosaccharide of the LPS of pseudomonads of biomedical importanceavailable for analysis and in complete characterization of thephosphate-free structure thereof. Furthermore, the invention hassucceeded in making it possible to isolate this previously unknown sugarfrom these bacteria and to analyze and quantify it by combined gaschromatography/mass spectrometry (GC-MS).

It has been possible to date to identify the novel7-O-carbamoylheptopyranose only in pseudomonads of RNA group 1(especially in the human-pathogenic P. aeruginosa and in P.fluorescens).

Furthermore, the novel sugar occurs in the LPS of all immunotypes of P.aeruoginosa (Fischer 2,7), investigated to date, irrespective of thestructure of the O chain. No exceptions to this rule are known as yet.The 7-O-carbamoylheptopyranose does not occur in the LPS of pseudomonadswhich are pathogenic to plants (for example P. plantarii), which are nowassigned to burkholderia, no longer to the group of pseudomonads.

Both the diagnostic and taxonomic significance of the7-O-carbamoylheptopyranose can be deduced from these findings. The7-O-carbamoylheptopyranose is likewise absent from all otherGram-negative bacteria investigated to date: Klebsiella pneumoniae, K25;Yersinia enterocolitica, mutant 490 M; Campylobacter jejuni RN 16 0:58;CCUG 10936; Proteus mirabilis, mutant R₄₅ ; Haemophilus influenzae, B,strain Eagan, Vibrio parahaemolyticus, serotype 012, Salmonellaminnesota, SF 1111, and E. coli O111 were negative for the7-O-carbamoylheptose in the screening process, which underlines onceagain the diagnostic importance of this newly discovered sugar.

Using the sugar according to the invention it has now become possiblefurther to improve the species-specific diagnosis of pseudomonads andtheir taxonomic classification.

Since it is known that monoclonal antibodies directed against epitopesof the inner and outer core regions are able also to recognize wild-typeLPS with analogous core oligosaccharides Di Padova, F. et al., Infect.Immun., 61 (1993) 3863-3872 and Rietschel, e.Th. et al., FASEB. J., 8(1994) 217-225!, it is possible on the basis of the present structureelucidation to define species-specific epitopes by means of monoclonalantibodies which cruccially simplify and improve the serodiagnosis ofthese human-pathogenic organisms. This serodiagnosis can at present becarried out only via the O-specific chain, with the known disadvantagesof lacking cross-specificities.

The pseudomonads employed in the process according to the invention,which occur in soil, water, waste water, on plants and in foodstuffs,are extremely well-known microorganisms which can also be obtained fromrecognized depositary authorities.

Depending on the conditions used for the methanolysis, it is possible bythe process according to the invention to produce either the heptosemonosaccharide or the heptose di- or oligosaccharide (see also Example3.1 hereinafter).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structural formula, the electron impact mass spectrum(1) and the Cl-- NH₃ ! mass spectrum (2) ofmethyl-2,3,4,6,7-penta-O-acetyl-D-glycero-α/β-D-manno-heptopyranose(compound No. 4).

FIG. 2 shows the structural formula, the electron impact mass spectrum(1) and the Cl-- NH₃ ! mass spectrum (2) ofmethyl-2,3,4,6,7-tetra-O-acetyl-7-O-carbamoyl-D-glycero-α/β-D-manno-heptopyranose(compound No. 5).

FIG. 3 shows the structural formula, the electron impact mass spectrum(1) and the Cl-- NH₃ ! mass spectrum (2) ofmethyl-7-O-(N,N-dimethylcarbamoyl)-2,3,4,6-tetra-O-methyl-D-glycero-.alpha./β-D-manno-heptopyranose(compound No. 3).

FIG. 4 shows the structural formula, the electron impact mass spectrum(1) and the Cl-- NH₃ ! mass spectrum (2) of methyl 3-O-7-O-(N,N-dimethylcarbamoyl)-2,3,4,6-tetra-O-methyl-heptopyranosyl!-2,4,6,7-tetra-O-methyl-heptopyranoside(compound No. 12).

FIG. 5 shows the structural formula and the electron impact massspectrum of methyl-7-O-N,N-di-(trideuteriomethyl)-carbamoyl)-2,3,4,6-tetra-O-trideuteriomethyl-D-glycero-α/β-D-manno-heptopyranose(compound No. 6).

FIG. 6 shows the structural formula, the electron impact mass spectrum(1) and the Cl-- NH₃ ! mass spectrum (2) ofmethyl-7-O-acetyl-2,3,4,6-tetra-O-methyl-D-glycero-α/β-D-manno-heptopyranose(compound No. 8).

FIG. 7 shows the structural formula, the electron impact mass spectrum(1) and the Cl-- NH₃ ! mass spectrum (2) of methyl3-O-acetyl-2,3,4,6-tetra-O-methyl-heptopyranosyl!-2,4,6,7-tetra-O-methyl-heptopyranoside(compound No. 14).

FIG. 8 shows the structural formula, the electron impact mass spectrum(1) and the Cl-- NH₃ ! mass spectrum (2) of1,5-di-O-acetyl-7-O-(N,N-dimethylcarbamoyl)-2,3,4,6-tetra-O-methyl-heptitol(compound No. 16).

FIG. 9 shows the structural formula and the electron impact massspectrum of 1,5-tri-O-acetyl-2,3,4,6-tetra-O-methyl-heptitol (compoundNo. 15).

FIG. 10 shows the structural formula, the electron impact mass spectrum(1) and the Cl-- NH₃ ! mass spectrum (2) of1,3,5-tri-O-acetyl-2,4,6,7-tetra-O-methyl-heptitol (compound No. 18).

FIG. 11 shows the structural formula and the electron impact massspectrum of1,3,5-tri-O-acetyl-7-O-(N,N-dimethylcarbamoyl)2,4,6-tri-O-methyl-heptitol(compound No. 17).

FIG. 12 shows the gas-liquid chromatogram (GC) of a standard ofper-O-acetylated-D-glycero-D-manno-heptitol (D,D-Hep) andL-glycero-D-manno-heptitol (L,D-Hep) (top) compared with the heptitolacetates from Hep I and Hep II isolated from the core oligosaccharide ofPAC605, after removal of the 7-O-carbamoyl group. Column: SPB-5™, 150°C.-3 min then 5°/min to 330° C.

FIG. 13 shows the structural formula and the ¹ H-NMR spectrum ofmethyl-7-O-carbamoyl-LαD-Hepp-(1→3)-LαD-Hepp (1→OMe) (compound No. 9α)(360 MHZ, D₂ O, room temperature).

FIG. 14 shows the structural formula and the ¹³ C--NMR spectrum ofmethyl-7-O-carbamoyl-LαD-Hepp-(1→3LαD-Hepp-(1→OMe) (compound No. 9α).(90 MHz, D₂ O, room temperature).

FIG. 15 shows the structural formula, the molecular formula and thelaser desorption mass spectrum ofmethyl-7-O-carbamoyl-LαD-Hepp-(1→30-LαD-Hepp-(1→OMe) (compound No. 9α).

EXAMPLE

The compounds mentioned hereinafter have the following structuralformulae in which Me=methyl and Ac=acetyl. ##STR4##

    ______________________________________                                        Compound No. 1:                                                               Compound No. 2:                                                                          R.sup.1 = R.sup.2 = H                                              Compound No. 3:                                                                           ##STR5##                                                          Compound No. 4:                                                                          R.sup.1 = R.sup.2 = Ac                                             Compound No. 5:                                                                           ##STR6##                                                          Compound No. 6:                                                                           ##STR7##                                                          Compound No. 7:                                                                          R.sup.1 = Me, R.sup.2 = H                                          Compound No. 8:                                                                          R.sup.1 = Me, R.sup.2 = Ac                                          ##STR8##                                                                     Compound No. 9:                                                                           ##STR9##                                                          Compound No. 10:                                                                         R.sup.1 = R.sup.2 = H                                              Compound No. 11:                                                                          ##STR10##                                                         Compound No. 12:                                                                          ##STR11##                                                         Compound No. 13:                                                                         R.sup.1 = Me, R.sup.2 = H                                          Compound No. 14:                                                                         R.sup.1 = Me, R.sup.2 = Ac                                          ##STR12##                                                                    Compound No. 15:                                                                         R.sup.1 = R.sup.4 = Ac, R.sup.2 = R.sup.3 = Me                     Compound No. 16:                                                                          ##STR13##                                                         Compound No. 17:                                                                          ##STR14##                                                         Compound No. 18:                                                                         R.sup.1 = R.sup.3 = Ac, R.sup.2 = R.sup.4 = Me                     ______________________________________                                    

Materials and Methods

Cultivation of bacteria and extraction of lipopolysaccharide Pseudomonasaeruginosa bacteria (PAO, PAC605, PAC557 and R5 (Habs 06) were obtainedfrom P. Meadow, University College, London, Great Britain Rowe, P. S. N.and Meadow, P. M., Eur. J. Biochem., 132, (1983) 329-337! and from J. S.Lam, University of Guelph, Ontario, Canada E. Altman, et al.,Biochemistry 1994, submitted, E. Altman et al., Int. Carb. Conference,Paris 1992, E. Altman, et al., 2nd IES Conference Vienna 1992!. The PAOmutant is deposited at the Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Braunschweig, under number DSM 1707. The rough formmutant PAC 605 was cultivated in a 100 l fermenter, and the other P.aeruginosa mutants were cultivated in 2 l shake flask cultures asdescribed Kulshin, V. A. et al., Eur. J. Biochem, 198(1991) 697-704!.Lipopolysaccharides from the strains P. aeruginosa Fischer immunotypes 2and 7 were obtained from Prof. B. Dmitriev, Moscow, P. aeruginosa170519, 170520 and FH-N-845 were obtained from Prof. E. S. Stanislavsky,Moscow and Pseudomonas plantarii DSM 7128 originated from the DSMDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Braunschweig and P. fluorescens ATCC 49271 was obtained from theAmerican Type Culture Collection, Rockville, Md., USA. All other LPScargen: Klebsiella pneumoniae, K 25; Yersinia enterocolitica, mutant 490M; Campylobacter jejuni RN 16 0:58; CCUG 10936; Proteus mirabilis,mutant R₄₅ ; Haemophilus influenzae, B, strain Eagan, Vibrioparahaemolyticus; serotype O12, Salmonella minnesota, SF 1111 (S form),and E. coli O111 (S form) originated from the LPS collection of theForschungsinstitut Borstel.

The acquired heptose was prepared as mono- or oligomer using the P.aeruginosa PAC 605 mutant. To do this, dried bacteria (38.1 g) werewashed with 1 liter each of ethanol, acetone and diethyl ether, and theethereal sediment was dried (28.6 g, 75%). The membrane fractionobtained in this way underwent enzymatic digestion with DNAse (frombovine pancreas, Boehringer, Mannheim) and RNAse (bovine pancreas,Sigma) and proteinase K (from Tritirachium album, Boehringer).Extraction took place by a modified phenol/chloroform/petroleum methodPCP II (5 parts of phenol, 5 parts of chloroform, 8 parts of petroleumether (boiling point 90°-100° C.) modified by Galanos et al., Galanos,C., Luderitz, O. and Westphal, O., J. Biochem., 9, (1969), 245-249!.Exhaustive dialysis of distilled water was then carried out. The yieldwas 2.2 g (6%, m/m). Extraction of all other pseudomonas strains tookplace in an analogous manner by the method of Galanos et al. with yieldsof between 1 and 5%.

Gas chromatography (GC)

The analysis by gas chromatography was carried out with a HewlettPackard gas chromatograph (model 5890, series II), equipped with a flameionization detector (FID). Hydrogen was used as carrier gas with acolumn inlet pressure of 0.08 MPa. An SPB-5™ column (30 m, 0.25 mm ID,0.25 μm film thickness, Supelco) which was operated with a temperaturegradient (150° C. for 3 min, then 5° C./min to 330° C.) served asseparation column. Evaluation was carried out using Hewlett PackardChemstation Software® on a Vectra 486/66U.

Combined gas-liquid chromatography/mass spectroscopy (GC-MS) analysis

The combined gas-liquid chromatography/mass spectroscopy (GC-MS) wascarried out with a Hewlett Packard HP 5989A MS engine which was equippedwith an HP 5890 series II gas chromatograph and a capillary column(HP-5®, 30 m, 0.25 mm, 0.25 μ, Hewlett Packard). Electron impact massspectra (EI-MS) were recorded with 70 eV. Ammonia was used as reactorgas for the chemical ionization mass spectrometry (CI-MS). The spectrawere evaluated with a Hewlett Packard Chemstation Software® on a Vectra486/66U.

NMR spectroscopy

¹ H-(360 MHz and ¹³ C-(90 MHz) NMR spectra of the mono- anddisaccharides were carried out using a Bruker NMR spectrometer (modelAM-360) at room temperature in D₂ O. The recording and processing ofone-dimensional data and spectra with homo- (¹ H,¹ H-COSY) andheteronuclear (¹ H,¹³ C-NMR) correlation took place using an ASPECT 3000computer (Bruker) with the standard Bruker software DISNMIR Version 8911 01.0.

Laser desorption mass apectrometry (LD-MS)

The laser desorption mass spectrometry (LD-MS) was carried out using aLamma 500 instrument (Leybol-Heraeus, Cologne) in the positive modewithout addition of cationizing salts as described B. Lindner et al.,in: Analytical Microbiology Methods, (1990), Plenum Press New York, A.Fox, S. L. Morgan, L. Larsson and G. Odhan (eds.), pp. 149-161!.

High pressure liquid chromatography (HPLC)

The high pressure liquid chromatography (HPLC) of themethylheptopyranose mono- and dimers (2α, 9α and 10α) was carried out ina DuPont system (pump 870, gradient controller 8800) on a Zorbax NH₂column (9×250 mm). The column was operated with a CH₃ CN--H₂ O gradientand a flow rate of 3.5 ml/min. The gradient for isolation of 9αconsisted of eluent A CH₃ CN--H₂ O 925:75 (v/v)! and eluent B CH₃ CN--H₂O 75:925 (v/v(!; 0% B-5 min-10% B-75 min-100% B-20 min-100% B!. Thegradient for purification of 10α was A: 92:8, (v/v)! and B: 50:50,(v/v)!; 0% B-5 min-0% B-75 min-100% B-20 min-100% B!. Sugar componentswere detected in the eluate using a chiral detector (No. 1000A, Knauer)and were collected in fractions (3.5 ml) (Foxy, Colora). Aliquots ofthese fractions were additionally tested by thin-layer chromatography(TLC). The retention times under the specified conditions were 21 min(9α) and 40 min (2α) and 99 min (10α).

Thin-layer analysis (TLC)

The thin-layer analysis took place on silica gel 60 F₂₅₄ (Merck)aluminum plates in chloroform/methanol/water (100:100:30), v/v). Thesugar derivatives were detected by spraying with 15% (v/v) H₂ SO₄ inEtOH and subsequently heating.

Derivatization and degradation reactions

1. Dephosphorylation of the LPS

LPS (20 mg) was stirred with aqueous HF (48%) at 4° C. in a Teflonvessel overnight. It was subsequently dialyzed exhaustively with wateruntil the pH reached neutrality. The inner dialyzate was lyophilized(12.2 mg, 61% m/m).

2. Liberation of the dephosphorylated core oligosaccharide

The dephosphorylated LPS (LPS-HF) was stirred in a sodium acetate buffersolution (0.1M NaOAc, pH 4.4) at room temperature for 8 h. Thedephosphorylated core oligosaccharide was isolated after removal oflipoid A by centrifugation (20,000×g, 30 min) and subsequent SephadexG-10 chromatography (2.5×120 cm) 7.2 mg, 59%, m/m, based on LPS-HF).

3. Production and derivatization of the methyl glycosides of the heptosemono- and oligomers

3.1 Production of the heptose methyl glycosides

The dephosphorylated LPS (42 mg) was converted by treatment withHCl/MeOH into the methyl glycoside mono- and dimers. The monomers wereproduced by hydrolyzing in 2M HCl/MeOH at 85° C. for 30 min, and theoligomers were produced by hydrolyzing in 0.5M HCl/MeOH at 85° C. for 30min, and were isolated by HPLC (Zorbax-NH₂). Yield of monomer (1, 1.4mg) and dimer (9α, 2.2 mg)

3.2. Derivatization of the heptose methyl glycosides

3.2.1 Peracetylation

The methyl glycosides (1.5 mg) produced as in 3.1 were derivatized withacetic anhydride/pyridine (Ac₂ O/pyridine) at 37° C. for 30 min andsubsequently analyzed by a GC-MS. Deuterium-labeled peracetylatedheptose derivatives were produced in an analogous manner byderivatization with (CD₃ CO)₂ O/pyridine.

3.2.2. Permethylation

The heptoses and the dephosphorylated core oligosaccharide (2 mg ofeach) were methylated by the method of Ciucanu and Kerek Ciucanu, C. andKerek, F., Carbohydr. Res., 131 (1984) 209-217!. The permethylatedheptose derivatives or oligosaccharides were purified on a silica gelcolumn (silica gel 60, 70-230 mesh, 0.5×5 cm). To do this, the columnswere equilibrated with chloroform and eluted with an increasingconcentration of methanol (M) in chloroform (C) (C-M 95:5; v/v).Trideuterio-labeled permethylated heptose derivatives were produced inan analogous manner by derivatizing with trideuterioiodomethane (CD₃ I)in place of iodomethane. The heptose was perethylated using ethyl iodidein place of iodomethane.

3.2.3 Reductive cleavage of the dimethylcarbamoyl radical using lithiumaluminum hydride

Systematic preliminary investigations on the permethylated7-O-carbamoylheptose revealed that the 7-O-carbamoyl substituent isretained as N,N-dimethyl-7-O-carbamoyl radical on the heptose afterdephosphorylation (48% HF), NaOAC hydrolysis, methanolysis andpermethylation. To liberate 7-O-carbamoylheptose, 2 mg of each of thepermethylated N,N-dimethyl-7-O-carbamoylheptose monomer 3 or dimer 12were dissolved in dry diethyl ether (1 ml) and stirred with 7.5 mg oflithium aluminum hydride (LiAlH₄) at room temperature for 30 min. Thesolvent was blown off in a stream of nitrogen, and excess LiAlH₄ wasdecomposed by dropwise addition of water. For further purification, theproduct was taken up in chloroform/methanol (98:2 , v/v) and, afterremoval of the excess salt by centrifugation, was dried (yields: 7, 0.9mg and 13, 0.7 mg).

3.2.4. Regioselective resubstitution of the decarbamoylated heptosemono- and dimer

The heptose derivatives 7 and 13 were heated with N,N-dimethylcarbamoylchloride (50 μl, SIGMA) in pyridine (500 μl) at 85° C. for 4 h.Subsequently, the solvent and reagent were removed under oil pump vacuumand the product was investigated by mass spectrometry. As an alternativeto the resubstitution using N,N-dimethylcarbamoyl chloride,derivatization was carred out with acetic anhydride/pyridine (3.2.1.)and the resulting products (8 and 14) were investigated by GLC-MS.

3.2.5. Methylation analysis of the heptose to determine the bindingratios in the heptose region

The methylation analysis was used initially to determine the site ofsubstitution of the carbamoyl radical. To do this, monosaccharide 3,disaccharide 12 and permethylated oligosaccharide were initiallyhydrolyzed with 0.5 ml of 2M trifluoroacetic acid (TFA, Merck) at 120°C. for 1 h. The N,N-dimethyl-7-O-carbamoyl radical is completelyeliminated under these conditions. Excess TFA was stripped off byevaporation with distilled water in a rotary evaporator three times. Thesample is subsequently reduced with sodium borodeuteride (NaBD₄) andacetylated, and the partially methylated deutero-reduced heptitolacetate is investigated by GC and GC-MS. 3.3. Assignment of thecarbamoylheptose to the L-glycero-D-manno-heptitol andD-glycero-D-manno-heptitol configuration

All of the heptoses hitherto found in the LPS have, without exception,the L-glycero-D-manno-heptopyranose or D-glycero-D-manno-heptopyranoseconfiguration. In order to be able to assign the 7-O-carbamoylheptose toone of these configurations, 4 mg of the LPS were dephosphorylated toLPS-HF. The 7-O-carbamoyl substituent was subsequently selectivelyeliminated under mild alkaline conditions (0.5M NaOH, room temperature,30 min). We showed by kinetic studies that the carbamoyl radical isselectively eliminated under these conditions without involving damageto the heptose. Subsequently, hydrolysis (0.1M HCl, 48 h, 100° C.),reduction (5% NaBH₄ in 10 mM NaOH, overnight, room temperature) andperacetylation (Ac₂ O/pyridine) were carried out. Relative allocation ofthe configuration took place by means of GC analysis by comparison withthe retention times of standard L-glycero-D-manno-heptitol andD-glycero-D-manno-heptitol which can easily be distinguished in the GLCas pairs of diastereomers. GC conditions: column SPB-5® (30 m, 0.25 mm,0.25μ, Supelco); gradient: 150° C. for 3 min, then 5°/min to 330° C.

3.4. Screening process for determining 7-O-carbamoylheptose in driedbacteria

Dried bacteria (0.2 g) are suspended in 0.5 ml of 48% aqueous HF (Merck)and stirred vigorously at 4° C. in a Teflon vessel equipped with aTeflon stirrer overnight. The retentate from subsequent exhaustivedialysis against distilled water was freeze-dried. The7-O-carbamoylheptose was then liberated from the LPS in the biomass bymethanolysis (0.5M HCl/MeOH, 85° C., 30 min) and methylated Ciucanu, C.and Kerek, F., Carbohydr. Res., 131, (1984) 209-217! and thepermethylated product was removed by centrifugation (3000 rpm, 15 min,Rotixa, Hettlich). The fatty acid methyl esters of lipoid A weresubsequently removed using a small column (silica gel 60, 70-230 mesh,0.5×30 mm, Merck) in 5 ml of ether/n-hexane (40:60), v/v) and then theproduct (3) was eluted with chloroform/methanol (C-M 95:5, v/v) andanalyzed and quantified by GLC-MS on an SPB-5® column. The retentiontime (t_(R)) of the permethylatedmethyl-7-O-(N,N-dimethylcarbamoyl)-L-glycero-α-D-manno-heptopyranoseanomeric structures (3α and 3β) was 17.76 min and 17.86 min respectivelyunder the stated conditions (SPB-5®).

3.5. Isolation of 7-O-carbamoyl-LαD-Hepp-(1→3)-LαD-Hepp-(1→OMe), 9α, forNMR analysis

To isolate and prepare pure disaccharides 9α, initially 50 mg of LPSwere hydrolyzed (0.1M HCl, 100° C., 85 min) and the precipitated lipoidA was removed by centrifugation. The supernatant was further purified onSephadex G-10 (2.5×120 cm, water) and subsequently lyophilized. Thiscore oligosaccharide was suspended in 4×0.5 ml aliquots in aqueous HF(48%, Merck) and vigorously stirred in Teflon vessels overnight. Dryingat the oil pump (6 h) was followed by hydrolysis (0.5M HCl/MeOH, 85° C.,30 min) and the amino-containing components GalN and Ala were removedfrom the hydrolyzate on an ion exchanger (Amberlite IR-120, H⁺ form).After the exchanger resin had been washed (50 ml), the H₂ O eluate wasfurther purified by HPLC. The purified disaccharide 9α, which was foundat R_(f) =0.54 in TLC, eluted in the HPLC with a retention time of 21min (yield of 9α, 1.23 mg).

3.6. Isolation of LαD-Hepp-(1→3)-LαD-Hepp-(1→OMe), 10α, andLαD-Hepp-(1→OMe), 2α, from the synthetic disaccharideLαD-Hepp-(1→3)-LαD-Hepp as reference substances for the NMR analysis

3.8 mg of the synthesized disaccharide3-O(L-glycero-α-D-manno-heptopyranosyl)-L-glycero-D-manno-heptopyranosePaulsen, H. et al., Liebigs. Ann. Chem., (1986), 675-686) were incubatedin 0.5M HCl/MeOH at 37° C. overnight. TLC analysis silica gel 60,chloroform/methanol/water (C-M-W) 100:100:30! revealed that, under theseconditions, besides the target structuremethyl-3-O-(7-O-carbamoyl-L-glycero-α-D-manno-heptopyranosyl)-L-glycero-α-D-manno-heptopyranosedisaccharide (10α) (R_(f) 0.40), there had also been partial formationof the monosaccharides methyl L-glycero-α-D-manno-heptopyranoside 2α(R_(f) 0.58) and methyl L-glycero-β-D-manno-heptopyranoside 2β (R_(f)0.52). It was possible to separate these individual components in asubsequent HPLC run (Zorbax NH₂, 9 250 cm, DuPont) with an increasinggradient from eluent A CH₃ CN--H₂ O 92:8 (v/v)! and eluent B CH₃ CN--H₂O 50:50 (v/v)! at a flow rate of 3.5 ml/min. It was possible in thiscase to separate the disaccharide target structure 10α from themonosaccharides 2α and 2β, with the α-anomeric methyl glycosides showinga positive, and the β-anomeric methylheptose showing a negative, signalin the chiral detector. Aliquots of these peaks were tested once againby TLC (C-M-W 100:100:30). The purified disaccharide 9α (TLC R_(f) =0.4)eluted in the HPLC with a retention time of 99 min, 2α (40 min, TLCR_(f) 0.58) and 2β (54 min, TLC R_(f) 0.52). The yields were: 10α, 0.81mg, 2α 1.7 mg, 2β0.70 mg.

4. Results and discussion

To characterize the heptose required according to the invention, firstlythe nature and position of the carbamoyl radical on the heptose weredetermined by means of various labeling experiments in the GC-MSanalysis. Then the linkage of the two heptoses found in the coreoligosaccharide of Pseudomonas aeruginosa PAC605 LPS and thesubstitution pattern of the carbamoyl radical in the disaccharide werelikewise determined by GC-MS. Finally, the heptose disaccharide wasisolated with the 7-O-carbamoyl radical on the heptose in intact formand underwent detailed analysis by one- and two-dimensional NMRspectrometry with ¹ H- and ¹³ C-homo- (¹ H,¹ H-COSY) and heteronuclear(¹ H,¹³ C-COSY) correlation. After the7-O-carbamoyl-L-glycero-D-manno-heptopyranose had been identified, ascreening process for determination thereof in bacteria was developedwithout previous extraction of the LPS.

4.1. GC-MS analysis of the 7-O-carbamoylheptose as per-O-acetylatedderivative 5

Methanolysis (85° C., 2M HCl/MeOH, 30 min) of isolated, dephosphorylatedcore oligosaccharide from P. aeruginosa PAC605 LPS, per-O-acetylationand subsequent GC-MS analysis showed two different heptoses. One elutedas methyl 2,3,4,6,7-penta-O-acetylheptopyranoside (4) with a shorterretention time (t_(R) =17.4 min) than a second, preivously unknownheptose derivative (t_(R) =21.5 min). In the CI-MS, both heptoses showeda pseudo-molecular ion peak M+NH₄ !⁺ =452 (4) and M+NH₄ !⁺ =453 (5)(FIGS. 1 and 2). The difference in mass of one atomic mass unit (AMU)between 4 and 5, and the significantly increased retention time can beexplained by the replacement of an acetyl radical (CO--CH₃) in 4 by acarbamoyl radical (CO--NH₂) in 5.

4.2. Identification of the carbamoyl group by GC-MS

To identify the carbamoyl group and determine its site of substitution,variously labeled derivatives of the unknown heptose were produced forthe GC-MS analysis (3-8) in order to be able to prove the structure withthis method.

4.2.1. GC-MS analysis of the permethylated 7-O-carbamoylheptose as mono-and disaccharide

After dephosphorylation and methanolysis, the methyl glycosides of theheptose were permethylated. It was possible in this case to identify thepermethylated 7O-carbamoylheptose both as methyl7-O-(N,N-dimethylcarbamoyl)-2,3,4,6-tetra-O-methylheptopyranoside (3)and as methyl 3-O-7-O-(N,N-dimethylcarbamoyl)-2,3,4,6-tetra-O-methylheptopyranosyl!-2,4,6,7-tetra-O-methylheptopyranoside(12) in the GC-MS analysis (FIG. 3 and FIG. 4). Moreover themonosaccharide 3 showed in the EI-MS a characteristic fragment ion withm/z=320 M--OCH₃ !⁺ and m/z=146 which can be explained by the massfragment HC=O⁺ Me--CO--N(CH₃)₂ ! which derives from cleavage of theC-5/C-6 bond. This made it probable in the first place that thecarbamoyl radical was a substituent on C-6 or C-7.

The disaccharide 12 (t_(R) =34.3 min) (FIG. 4) showed in the EI-MS amass fragment with m/z=320, which indicated that the carbamoylsubstituent must have been located on the second, nonreducing heptose(Hep II). This interpretation is consistent with the existence of a massfragment with m/z=263, which was assigned to the permethylated, reducingheptopyranose (Hep I). The results of the CI-MS support thisinterpretation ( M+NH₄ !⁺ =617, FIG. 4). It was possibly byperethylation in place of permethylation to convert monosaccharide 3 anddisaccharide 12 into the relevant perethylated derivatives, it beingpossible to introduce 6 and 10, respectively, ethyl groups in place ofthe methyl group into 3 and 12, which was detectable in the MS analysisby a mass increment of Δm/z=14 AMU per ethyl radical introduced(detailed spectra not given here).

4.2.2. Labeling experiments and GC-MS analysis on 7-O-carbamoylheptosemono- and disaccharide

Methylation with iodomethane-d₁ in place of iodomethane allowed compound6 to be identified as monosaccharide in the GC-MS analysis (FIG. 5).Once again, the characteristic M--OMe!⁺ fragment with m/z=338 wasobserved, which corresponds to the fragment with m/z=320 in 3·m/z=155was found analogously and was derived from cleavage of the C-5/C-6 bond.It was thus possible unambiguously to locate the carbamoyl group inposition C-7 of the heptose from the fragment with m/z=107CH═O--CO--N(CD₃)₂ ! which corresponds to the nondiagnostic m/z=101CH═O--CO--N(CH₃)₂ ! in 3.

4.2.3. Reductive cleavage of the 7-O-carbamoyl radical using LiAlH₄

Compounds 7 and 13 were obtained by a mild and selective elimination ofthe permethylated carbamoyl radical from 3 and 12 respectively usingLiAlH₄ in ether, and these have a free primary hydroxyl group inposition 7 or 7' in place of the permethylated 7-O-carbamoyl group.Regioselective resubstitution of these free hydroxyl groups usingN,N-dimethylcarbamoyl chloride in pyridine afforded the startingcompounds 3 and 12 which did not differ in respect of retention time andmass fragmentation (EI-MS, CI-MS) from the initial substances. Thisprovided a further indication of the postulated structure.

O-acetylation of 7 and 13 results in 8 and 14 respectively (FIG. 6 andFIG. 7), both of which show the charactertistic fragment with m/z=117CHOMe--CH₂ --OAc!⁺ which is derived from cleavage of the C-5/C-6 bond inthe heptose. It was likewise possible to conclude from the fact that themass fragment with m/z=263 was not changed either in the disaccharide 12or in 14 that only the terminal heptose (Hep II) was substituted by thecarbamoyl radical and that substitution on Hep I is precluded.

4.3. Methylation analysis of the heptose region in the coreoligosaccharide

Monosaccharide 3 (3.5 mg) was hydrolyzed (2M TFA, 1 h, 120° C.), reduced(NaBD₄) and per-O-acetylated. We found that under these conditions the7-O-carbamoyl radical is not quantitatively eliminated, and both1,5,7-tri-O-acetyl-2,3,4,6-tetra-O-methylheptitol 15 (FIG. 9) and1,5-di-O-acetyl-7-O-N,N-dimethylcarbamoyl!-2,3,4,6-tetra-O-methylheptitol 16 are obtained(FIG. 8). The partially methylated heptitol acetate 15 (t_(R) =15.1 min,FIG. 9) showed in the CI-MS M+NH₄ !⁺ m/z=413 and M+H!⁺ m/z=396 and inthe EI-MS fragments with m/z=102 (162-60), 118 and 162 fromdeuterium-reduced end, and with m/z=117, 223 and 277 from the unreducedend. The partially methylated heptitol 16 (t_(R) =19.7 min) showed inthe CI-MS (FIG. 8) M+NH₄ !⁺ m/z=442 and M+H!⁺ m/z=425 and in the EI-MSfragments with m/z=102 (162-60), 118, 162 from the deuterium-reducedend, and with m/z=102, 146, 262, 306 from the unreduced end. These massfragments from the partially methylated heptitol acetates provided afurther indication of 7-substitution by the carbamoyl radical.

The linkage of the two heptoses (HepII-Hep I) together was determined bymethylation analysis of the permethylated disaccharide 12.1,3,5,-Tri-O-acetyl-2,4,6,7-tetra-O-methylheptitol (18, t_(R) =15.1 min,FIG. 10) was obtained, from which it was possible to determine thelinkage of the heptoses together as Hepp-(1→3)-Hepp. The mass fragments(EI-MS) were at m/z=118, 234, 350 and were identified as fragments fromthe reducing end (FIG. 10). The data from the CI-MS analysis, M+NH₄ !⁺with m/z=413 and M+H!⁺ with m/z=396 are consistent with thisinterpretation.

On the other hand, carrying out the methylation analysis described aboveon the intact core oligosaccharide in place of the disaccharide revealeda1,3,5-tri-O-acetyl-7-O-(N,N-dimethylcarbamoyl)-2,4,6-tri-O-methylheptitol17 with a retention time of 24.5 min. The existence of a 3-O-acetylated7-O-carbamoylheptitol in 17 can be explained by substitution of the GalNAla! residue in position 3 of Hep II in the intact core oligosaccharide.This result is consistent with data obtained earlier by Rowe and MeadowRowe, P. S. N. and Meadow, P. M., Eur. J. Biochem., 132 (1983) 329-337!and E. Altman et al. (E. Altman et al., Biochemistry 1994, submitted forpublication; E. Altman et al., Int. Carb. Conference, Paris 1992,Abstract Book C161, p. 626; H. Masoud et al., 2nd Conference of theInternational Endotoxin Society (IES), Vienna 1992, 69, p. 55), who haddetermined the site of substitution of GalN-Ala and the linkage of theheptoses with one another likewise as GalpNAla!-(1→3)-LαD-Hepp-(1→3)-LαD-Hepp.

4.4. Assignment of the 7-O-carbamoylheptose to the L-glycero-D-manno orD-glycero-D-manno configuration

Measured first in the GLC analysis (SPB-5®) was a standard whichcontained both peracetylated D-glycero-D-manno-heptitol (D,D-Hep-ol) andL-glycero-D-manno-heptitol (L,D-Hep-ol) (t_(R) =21.36 min, D,D-Hep:t_(R)=21.89 min, L,D-Hep). After the 7-O-carbamoyl radical on Hep II had beenselectively removed from the core oligosaccharide of P. aeruginosaPAC605 by mild alkaline hydrolysis, the heptitol acetates prepared fromHep I and Hep II in this was were investigated in GC analysis. Itemerged that only L-glycero-D-manno-heptitol acetate was detectable,t_(R) =21.89 min, which corresponds to L,D-Hep-ol (FIG. 12). Thisexperiment and the fact that the heptose remains intact on eliminationof the carbamoyl substituent made it probable that the7-O-carbamoylheptopyranose (Hep II) has the L-glycero-D-mannoheptoseconfiguration (compare also NMR analysis).

4.5 NMR analysis of themethyl-3-O-(7O-carbamoyl-L-glycero-α-D-manno-heptopyranosyl)-L-glycero-α-D-mannoheptopyranose9α

4.5.1. ¹ H-NMR analysis

The disaccharide3-O-(7-O-carbamoyl-L-glycero-α-D-manno-heptopyranoysl)-L-glycero-.alpha.-D-manno-heptopyranose9α was isolated after mild methanolysis from the dephosphorylated coreoligosaccharide and was purified by HPLC. The results of the proton NMRanalysis are depicted in FIG. 13 and Table 1. It is significant thatthere is a low-field shift of the signals for H-7'a and H-7'b by about0.3 ppm (4.085 and 4.154 ppm) in 7-O-carbamoylheptose compared with theanalog signals for H-7a and H-7b in the unsubstituted heptose (3.7276and 3.751 ppm respectively). Substitution in position 3 of Hep I ismanifested only by a low-field shift of about 0.12 ppm compared with theα-anomeric methyl L-glycero-D-manno-heptopyranoside 2α. All the othersignals agree well with the synthetic disaccharidemethyl-3-O-(L-glycero-α-D-manno-heptopyranosyl)-L-glycero-α-D-manno-heptopyranose(10α). The good agreement of the signals is a further indication of theL-glycero-α-D-manno configuration found in the GC analysis.

4.5.2 ¹³ C-NMR analysis of disaccharide 9α

The results of the ¹³ C-NMR analysis derived from the spectra withheteronuclear correlation for the disaccharide3-O-(7-O-carbamoyl-L-glycero-α-D-manno-heptopyranosyl)-L-glycero-.alpha.-D-manno-heptopyranose(9α) are to be found in FIG. 14 and Table 2. Comparison with thesynthetic disaccharidemethyl-3-O-(L-glycero-α-D-manno-heptopyranosyl)-L-glycero-α-D-manno-heptopyranose(10α) and the synthetic monosaccharide methylL-glycero-α-D-manno-heptopyranoside (2α) made it possible to deduce theindividual structural features of the heptose (7-O-carbamoylsubstitution, glycosidic linkage and configuration). The carbonylCI--NH₂ signal at 159.86 ppm is particularly noteworthy and agrees wellwith an analog signal (159.6 ppm) for a 6-O-carbamoyl-GlcN(Me: 18:0)-Rfrom Azorhizobium caulinodans which was recently described (Mergaert, P.et al., Proc. Natl. Acad. Sci. USA, 90 (1993) 1151-1555). The low-fieldshift of the signal for C-7' in 9α is only about 2.2 ppm by comparisonwith the unsubstituted compound 10α, which is not unusual for glycosidicsubstituents on primary hydroxyl groups. On the other hand, thelow-field shift of C-3 on Hep I (71.35 vs. 77.89 ppm) is significant,which again confirms the Hep-(1→3)-Hep substitution in the disaccharide.All the other signals agree well with the disaccharide 10α. The goodagreement of the ¹³ C signals with the synthetic reference compounds 2αand 10α is a further indication of the L-glycero-α-D-manno configurationfound in both heptoses.

4.6 Laser desorption mass analysis (LD-MS) of themethyl-3-O-(7-O-carbamoyl-L-glycero-α-D-manno-heptopyranosyl)-L-glycero-α-D-manno-heptopyranose9α

The result of the laser desportion mass analysis of compound 9α isdepicted in FIG. 15. The molecular weight of themethyl-3-O-(7-O-carbamoyl-L-glycero-α-D-manno-heptopyranosyl)-L-glycero-α-D-heptopyranose(9α) was calculated for the molecular formula C₁₈ O₁₄ H₂₉ N as 459.40.The LD-MS spectrum showed the purified disaccharide 9α as a uniquepseudomolecular peak with a molar mass of M+Na!⁺ =459+23=482 and is thusin excellent agreement with the calculated structure shown.

                  TABLE 1                                                         ______________________________________                                        Chemical shift, assignment and coupling constants for the signals in the      .sup.1 H-NMR spectrum of methyl-7-O-carbamoyl-LαD-Hepp-(1→3)-    LαD-                                                                    Hepp-(1→OMe) 9α compared with the synthetic                      LαD-Hepp-(1→3)-                                                  LαD-Hepp-(1→OMe) 10α and LαD-Hepp-(1→OMe)     2α reference                                                            compounds (360 MHz, D.sub.2 O, room temperature).                             ______________________________________                                        7-O-carbamoyl-LαD-Hepp-(1→                                       9α           10α                                                       δ (ppm)                                                                            J (Hz)     δ (ppm)                                                                       J (Hz)                                       ______________________________________                                        H-1' 5.197      1.3        5.150 1.7                                          H-2' 4.093      2.6        4.071 3.1                                          H-3' 3.913      6.2         3.894*                                            H-4' 3.703      5.7         3.894*                                                                             9.8                                          H-5' 3.709      1.3        3.705 1.9                                          H-6' 4.228      7.9        4.052 5.4                                          H-7'a                                                                              4.085      10.5       3.729 11.9                                         H-7'b                                                                              4.154      6.2        3.760 6.8                                          ______________________________________                                        →3)-LαD-Hepp-(1→OMe)                                                                 2α                                           ______________________________________                                        H-1  4.717      1.3        4.747 1.9   4.737                                                                              1.5                               H-2  3.983      3.5        4.031 3.2   3.895                                                                              3.4                               H-3  3.906      10.3       3.842 9.9   3.729                                                                              9.8                               H-4  3.983      9.7        3.967 10.0  3.825                                                                              10.2                              H-5  3.600      1.5        3.604 1.6   3.544                                                                              1.9                               H-6  4.055      6.8        4.048 5.8   4.015                                                                              5.8                               H-7a 3.727      11.6       3.662 11.4  3.685                                                                              11.1                              H-7b 3.751      6.9        3.741 7.4   3.731                                                                              7.3                               OMe  3.370                 3.389       3.380                                  ______________________________________                                         *unresolved multiplet                                                    

                                      TABLE 2                                     __________________________________________________________________________    Chemical shift and assignment of the .sup.13 C-NMR signals of                 7-O-carbamoyl-LαD-                                                      Hepp-(1→3)-LαD-Hepp-(1→OMe) 9α, compared with       synthetic reference compounds                                                 LαD-Hepp-(1→3)-LαD-Hepp-(1→OMe) 10α and       LαD-Hepp-(1→OMe) 2α.                                       __________________________________________________________________________             α (ppm)                                                                 9α         10α                                           C atom   7-O-carbamoyl-LαD-Hepp-(1→                                                        LαD-Hepp-(1→                           __________________________________________________________________________    C-1'     103.06           103.30                                              C-2'     70.83            70.91                                               C-3'     71.33            71.35                                               C-4'     66.89            66.70                                               C-5'     72.09            72.63                                               C-6'     66.69            69.51                                               C-7'     65.90            63.68                                               --O--CO--NH.sub.2                                                                      159.86                                                               __________________________________________________________________________                               2α                                              →3)-LαD-Hepp-OMe                                                             →3)-LαD-Hepp-OMe                                                             LαD-Hepp-OMe                                 __________________________________________________________________________    C-1                                                                              101.77      101.75      103.30                                             C-2                                                                              70.70       70.56       70.91                                              C-3                                                                              77.89       70.09       71.35                                              C-4                                                                              66.73       66.45       66.64                                              C-5                                                                              72.26       72.04       72.04                                              C-6                                                                              69.48       69.64       69.51                                              C-7                                                                              63.73       63.68       63.68                                              __________________________________________________________________________     *(D.sub.2 O, 90.556 MHz, ppm relative to internal acetonitrile 1.700 ppm)

                  TABLE 3                                                         ______________________________________                                        Identification of the 7-O-carbamoylheptose in the core                        oligosaccharide from various Gram-negative bacteria                           7-O-carbamoylheptopyranose                                                    ______________________________________                                        1. Pseudomonadaceae (old classification)                                      1.1 Rough form mutants                                                        Pseudomons aeruginosa PAC605                                                                            +                                                   P. aeruginosa PAC 557     +                                                   P. aeruginosa PAC1R       +                                                   P. aeruginosa RS (Habs 6) +                                                   1.2. Smooth form bacteria                                                     P. aeruginosa Fischer 2 immunotype                                                                      +                                                   P. aeruginosa Fischer 7 immunotype                                                                      +                                                   P. aeruginosa 170519      +                                                   P. aeruginosa 170520      +                                                   P. aeruginosa FH-N-845    +                                                   P. fluorescens ATCC 49271 +                                                   Pseudomonas plantarii DSM 712B                                                                          -                                                   2. Non-Pseudamonadaceae                                                       Klebsiella pneumoniae, K 25                                                                             -                                                   Yersinis enterocolitica, mutant 490 M                                                                   -                                                   Campylobacter jejuni RN16 O:58, CCUG 10936                                                              -                                                   Proteus mirabilis mutant R.sub.45                                                                       -                                                   Haemophilus influenzae, wild-type, Eagan strain                                                         -                                                   Vibrio parahaemolyticus, serotype O12                                                                   -                                                   Salmonella minnesota SF 1111 (S form)                                                                   -                                                   Escherichia coli O111 (S form)                                                                          -                                                   ______________________________________                                    

We claim:
 1. 7-O-Carbamoylheptose derivatives of the formula ##STR15##wherein R is R¹ or a group of the formula (II) ##STR16## wherein R¹ is ahydrogen atom, a methyl group or a linker substituent suitable forcovalent coupling.
 2. 7O-Carbamoylheptose derivatives according to claim1 wherein the linker substituent is a cysteamine residue, an allyl groupor a straight-chain or branched-chain C₁₋₁₈ alkyl group which isunsubstituted or substituted with a hydroxyl, amino, acyl, carboxyl orallyl group.
 3. 7O-Carbamoylheptose derivatives according to claim 1wherein R¹ is a hydrogen atom or a methyl group.
 4. Compositions fortreating Pseudomonas infections comprising an effective amount of atleast one carbamoylheptose derivative according to claim 1 in apharmaceutically acceptable carrier.
 5. A method for treatingPseudomonas infections comprising administering to a patient in needthereof an effective amount of at least one carbamoyheptose derivativeaccording to claim
 1. 6. A process for preparation of7O-carbamoylheptose derivatives of the formula ##STR17## wherein R is R¹or a group of the formula (II) ##STR18## wherein R¹ is a hydrogen atom,a methyl group or a linker substituent suitable for covalent coupling;comprising:(a) treating intact Gram-negative bacteria with hydrofluoricacid; (b) hydrolyzing or metholyzing the treated bacteria to liberateheptose derivatives; and (c) permethylating the heptose derivatives.